A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.
Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.
Neural crest cells (NCCs) are a group of stem cells during vertebrate embryogenesis with a remarkable ability to migrate and contribute to the development of various organs and tissues. NCCs can differentiate into different cell types, including sensory neurons, cartilage, bone, melanocytes, and smooth muscle cells, depending on the location of axial origin and the local environmental guidance of the NCC1,2. With the ability to differentiate into a wide array of cell types, genetic abnormalities that cause dysregulation at any stage of neural crest (NC) development can lead to numerous congenital diseases
1. Hydrogel preparation
NOTE: All steps must be performed in a cell culture hood that has been disinfected with ethanol and ultraviolet (UV)-sterilized before use to maintain sterility. Tools, such as tweezers and pipettes, must be sprayed with ethanol. Buffer solutions must also be sterile-filtered.
Hydrogel preparation and stiffness assessment through AFM and the Hertz model
Here, a detailed protocol is provided to generate polyacrylamide hydrogels of varying stiffness by regulating the ratio of acrylamide and bis-acrylamide. However, the polyacrylamide hydrogels are not ready for the adhesion of cells due to the lack of ECM proteins. Thus, sulfo-SANPAH, acting as a linker, covalently binds to the hydrogels and reacts with the primary amines of ECM proteins to allow the adhesion of ECM protei.......
The goal of the current study is to provide an effective and efficient in vitro system to better understand the impact of mechanical signals in NCCs. In addition to following the step-by-step protocol mentioned above, researchers need to keep in mind that the cell culture of O9-1 NCCs is affected by the type of glass coverslips used to prepare hydrogels. For instance, it was noted that cells seeded on a specific type of glass coverslip (see the Table of Materials) survived and proliferated .......
We thank Dr. Ana-Maria Zaske, operator of Atomic Force Microscope-UT Core facility at the University of Texas Health Sciences Center, for the contributed expertise in AFM in this project. We also thank the funding sources from the National Institutes of Health (K01DE026561, R03DE025873, R01DE029014, R56HL142704, and R01HL142704 to J. Wang).
....Name | Company | Catalog Number | Comments |
12 mm #1 Corning 0211 Glass Coverslip | Chemglass Life Sciences | CLS-1763-012 | |
2% Bis-Acrylamide | Sigma Aldrich | M1533 | |
24-well plate | Greiner Bio-one | 662165 | |
25 mm #1 Corning 0211 Glass Coverslip | Chemglass Life Sciences | CLS-1763-025 | |
3-aminopropyl triethoxysilane (APTS) | Sigma Aldrich | A3648 | |
4-well cell culture plate | Thermo Scientific | 179830 | |
4% Paraformaldehyde | Sigma Aldrich | J61899-AP | |
40% Acrylamide | Sigma Aldrich | A4058 | |
50% glutaraldehyde | Sigma Aldrich | G7651 | |
6-well cell culture plate | Greiner Bio-one | 657160 | |
AFM cantilever (spherical bead) | Novascan | ||
AFM software | Catalyst NanoScope | Model: 8.15 SR3R1 | |
Alexa Fluor 488 Phalloidin | Thermo Fisher | A12379 | |
Ammonium Persulfate (APS) | Sigma Aldrich | 248614 | Powder |
anti-AP-2α Antibody | Santa Cruz | sc-12726 | |
anti-Vinculin antibody | Abcam | ab129002 | |
Atomic Force Microscopy (AFM) Bioscope Catalyst | Bruker Corporation | ||
Collagen type I (100mg) | Corning | 354236 | |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher | D1306 | |
Dichloromethylsilane (DCMS) | Sigma Aldrich | 440272 | |
Donkey serum | Sigma Aldrich | D9663 | |
Dulbecco's Modified Eagle Medium (DMEM) | Corning | 10-017-CV | |
Fetal bovine serum (FBS) | Corning | 35-010-CV | |
Fluorescence microscope | Leica | Model DMi8 | |
Fluoromount-G mounting medium | SouthernBiotech | 0100-35 | |
HEPES | Sigma Aldrich | H3375 | Powder |
Horse serum | Corning | 35-030-CI | |
iScript Reverse Transcription Supermix | Bio-Rad | 1708841 | |
Penicillin-Streptomycin antibiotic | Thermo Fisher | 15140148 | |
RNeasy micro kit | Qiagen | 74004 | |
Sterile 1x PBS | Hyclone | SH30256.02 | |
Sterile deionized water | Hardy Diagnostics | U284 | |
sulfo-SANPAH | Thermo Fisher | 22589 | |
SYBR green | Applied Biosystems | 4472908 | |
TEMED | Sigma Aldrich | T9281 | |
Triton X-100 | Sigma Aldrich | X100 | |
Tween 20 | Sigma Aldrich | P9416 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved